Detection of herpes simplex virus types 1 and 2 by nucleic acid amplification

ABSTRACT

The present invention relates to a method of detecting the presence or absence of herpes simplex virus (HSV) in a sample based on amplifying a portion of the Glycoprotein G(US4) gene of HSV and detecting the presence of the amplified nucleic acid using primers and detector primers as described herewith. The method of the invention further identifies the type of HSV, either HSV-1 or HSV-2, in a sample. Also encompassed by the invention is a kit comprising the primers and detector primers which may be used with the amplification method described herewith.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. patent applicationSer. No. 10/832,120, filed Apr. 26, 2004 under 35 U.S.C. §120 andPCT/US2004/012766, filed Apr. 26, 2004, both of which claims priority toU.S. Provisional Application No. 60/465,458, filed Apr. 25, 2003 under35 U.S.C. §119(e), the entirety of all of which are incorporated hereinby reference as if fully set forth herein.

FIELD OF THE INVENTION

The present invention relates to diagnostic methods and nucleic acidsequences for identifying Herpes Simplex Virus (HSV) by nucleic acidamplification methods.

BACKGROUND OF THE INVENTION

Herpes Simplex is an enveloped double-stranded DNA virus that isresponsible for primary and recurrent infections in humans and isrelated to the viruses that cause infectious mononucleosis (Epstein-BarrVirus), chicken pox and shingles (Varicella Zoster Virus). Symptoms ofHerpes Simplex Virus (HSV) infections include an eruption of tinyblisters on the skin or mucous membranes. After the eruption of blisterssubsides, the virus remains in a dormant (latent) state inside the groupof nerve cells (ganglia) that supply the nerve fibers to the infectedarea. Periodically, the virus reactivates, begins growing again, andtravels through the nerve fibers back to the skin, thereby causingeruptions of blisters in the same area of skin as the earlier infection.Sometimes the virus may be present on the skin or mucous membranes evenwhen there is no obvious blister. Herpes Simplex Virus (HSV) isclassified into two types, HSV-1 and HSV-2. The complete genomes ofhuman HSV-1 and HSV-2 have been sequenced (see, for example, NCBIAccession Nos. X14112 and Z86099, respectively).

HSV has been shown to contribute to or cause a variety of disorders,including blindness and encephalitis. Besides causing local outbreaks,HSV-1 and HSV-2 are associated with encephalitis. The pathophysiology ofthis encephalitis is poorly understood in humans Animal models suggestthat the virus enters the central nervous system through peripheralnerves and causes inflammation of the brain. HSV-1 is the more commoncause of adult encephalitis. HSV-2 is the more common cause of newbornencephalitis, which is associated with maternal genital infections.HSV-2 is one of the most common sexually transmitted diseases insociety. HSV-related encephalitis has the highest fatality rate of allthe types of encephalitis with an annual incidence of 1 to 4 permillion. HSV encephalitis affects people of all ages and at any time ofthe year. In adults, HSV-related encephalitis is thought to be due to areactivation of a latent virus. Symptoms may include fever, headaches,seizures, an altered level of consciousness and personality changes. Thesimilarity of these symptoms to other maladies makes clinical diagnosisdifficult. If left untreated, the mortality rate for herpes simplexencephalitis (HSE) is as high as seventy percent, compared with as lowas nineteen percent among those who receive treatment. Of the treatedpatients, approximately thirty-eight percent are reported to eventuallyreturn to normal function. It is, therefore, very important to be ableto diagnose HSV infection at an early stage.

The diagnosis of HSV infection is commonly performed using cell cultureon appropriate clinical specimens. However, the ability to isolate HSVin cell culture is reduced in old lesions, in the presence of a hostimmune response and in episodes of reactivation. Serologic diagnosis,particularly of HSV in cerebrospinal fluid (CSF), is not sufficientlysensitive or specific, and takes too much time to be of use in decisionsinvolving choices for early therapeutic intervention of encephalitis.HSV is rarely detected in cerebral spinal fluid using cell culture, withonly four percent of the cases being culture-positive. Serologicalmethods are also inadequate for diagnosis of HSE due to delay betweentwo and three weeks in appearance of antibody response after initialinfection. The “gold standard” method of diagnosis involving brainbiopsies is invasive and controversial with significant risk oflong-term morbidity. Alternate techniques such as Computer-AssistedTomography and Magnetic Resonance Imaging are not specific and lacksensitivity as diagnostic tools.

At the present time, immunological methods for detection of HSV areunreliable and difficult to perform. Molecular methods of detectionoffer the potential for enhanced sensitivity and faster time to resultthan is possible by conventional means. There are instances in whichrapid, sensitive, and specific diagnosis of HSV disease is imperative.There is therefore, a clinical need to develop a rapid and sensitivetool to aid in the diagnosis of HSV. There also remains a need for atool for the typing of the HSV infection. Rapid identification of thespecific etiological agent involved in a viral infection providesinformation which can be used to determine appropriate therapy within ashort period of time.

SUMMARY OF THE INVENTION

The present invention relates to methods and compositions fordetermining the presence of Herpes Simplex Virus (HSV), specificallyHerpes Simplex Virus type 1 (HSV-1) or type 2 (HSV-2) in mammals. Themethod involves using primers to amplify and detect Herpes Simplex Virustarget sequence. One embodiment uses the amplification technique ofStrand Displacement Amplification (SDA).

The nucleic acid primers of the invention uniquely amplify the targetsequence in HSV-1 or HSV-2, thereby allowing sensitive detection andtype-identification of HSV. The present invention is also directed to aStrand Displacement Amplification (SDA) based method for the detectionof HSV that involves real-time detection using a universal fluorescentenergy transfer probe. The probes and primers of the present inventionprovide a direct, rapid, and sensitive detection of HSV nucleic acidsand therefore offer an attractive alternative to immunological assays.

The probes and primers of the invention may be used after culture of thesample as a means for confirming the identity of the cultured organism.Alternatively, they may be used prior to culture or in place of culturefor detection and identification of HSV nucleic acids using knownamplification methods. The inventive probes, primers, and compositionsand assay methods using the probes, primers, and compositions, provide ameans for rapidly discriminating between the nucleic acid targetsequences of HSV-1 and HSV-2, allowing the practitioner to identify,diagnose, and treat the HSV type without resorting to the time-consumingimmunological and biochemical procedures typically relied upon.

BRIEF DESCRIPTION OF THE DRAWINGS

The various objects, advantages and novel features of the presentinvention will be readily understood from the following detaileddescription when read in conjunction with the appended drawings inwhich:

FIG. 1 shows a consensus sequence (SEQ ID NO: 1) of the Glycoprotein G(US4) gene of Herpes Simplex Virus Type 1 (HSV-1).

FIG. 2 is a map showing a portion of the genomic sequence of the HSV-1target region (SEQ ID NO: 2) and the location of primers, bumpers, andadapters designed for specific detection of HSV-1 DNA.

FIG. 3 is a graph showing “MOTA” expression of results.

FIG. 4 is a graph showing the “PAT” algorithm used with the BD Probelec™ET System.

FIG. 5 depicts the analytical sensitivity of the SDA method on dilutionsof various HSV-1 strains.

FIG. 6 is a consensus sequence (SEQ ID NO: 3) of a fragment of theGlycoprotein G (US4) gene of Herpes Simplex Virus Type 2 (HSV-2).

FIG. 7 is a map showing the genomic sequence of the HSV-2 target region(SEQ ID NO: 4) and the location of primers, bumpers, and adaptersdesigned for specific detection of HSV-2 DNA.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated and purified nucleic acids,polynucleotides, amplification primers and assay probes which exhibitHerpes Simplex Virus (HSV) type specificity in nucleic acidamplification reactions. Also provided are methods for detecting andidentifying HSV nucleic acids using the probes and primers of theinvention.

One embodiment of the present invention relates to an amplificationmethod for detecting the presence of a target nucleic acid sequenceusing one or more amplification primers having a target bindingsequence, producing an amplified target sequence, and detecting thetarget sequence. Non-limiting examples of amplification methods includePolymerase Chain Reaction (PCR; see Saiki et al., 1985, Science230:1350-1354, herein incorporated by reference), Ligase Chain Reaction(LCR; see Wu et al., 1989, Genomics 4:560-569; Barringer et al., 1990,Gene 89:117-122; Barany, 1991, Proc. Natl. Acad. Sci. USA 88:189-193,all of which are incorporated herein by reference), in situhybridization, Transcription Mediated Amplification (TMA; see Kwoh etal., 1989, Proc. Natl. Acad. Sci. USA 86:1173-1177, herein incorporatedby reference), Self-Sustaining Sequence Replication (3SR; see Guatelliet al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878, hereinincorporated by reference), Rolling Circle Amplification (RCA), NucleicAcid Sequence Based Amplification (NASBA), Qβ replicase system (Lizardiet al., 1988, BioTechnology 6:1197-1202, herein incorporated byreference) and Strand Displacement Amplification (SDA; see Walker etal., 1992, Proc. Natl. Acad. Sci. USA 89:392-396; Walker et al., 1992,Nuc. Acids. Res. 20:1691-1696; and EP 0 497 272, all of which areincorporated herein by reference)) including thermophilic SDA (tSDA).

Another embodiment of the present invention relates to an isothermalStrand Displacement Amplification (SDA) method for detecting thepresence of HSV nucleic acid sequences in a sample by exponentialamplification of the HSV target sequence. In a further embodiment, SDAis performed at about 52° C. as described in U.S. Pat. No. 5,648,211using a selected detector primer to detect a target during amplificationas described in U.S. Pat. Nos. 5,919,630; 5,928,869; 5,958,700; and6,261,785, all of which are hereby incorporated by reference. As typicalwith SDA, reagents, primers, enzymes, such as restriction enzymes andpolymerase, and other components are added to a reaction microwell,container, or receptacle. SDA amplifies a specific DNA sequence from asample, where once all the components are mixed together, the reactioncontinues until a critical component is exhausted. In contrast to thepolymerase chain reaction (PCR), SDA is an isothermal reaction processsuch that, once the reaction is initiated, there is no external controlover the progress of the reaction.

The SDA method of the present invention requires at least two HSVamplification primers and two bumper primers to initiate theamplification method. The amplification primers are designed to behighly specific for HSV-1 or HSV-2. The SDA method involves concurrentamplification reactions in a mixture and does not require separatephases or cycles for temperature cycling as is necessary in a PCRamplification method. A further advantage of the SDA of the presentinvention is exponential amplification. The steps of DNA polymeraseextension, nicking, displacement, and regeneration of the nick siteresult in displaced single-stranded molecules with partial restrictionenzyme sites (e.g., BsoBI sites) at either end which then circulate andare captured by amplification primers, thereby exponentially amplifyingthe HSV target sequence. The SDA method also provides an improvedworkflow, especially for high-throughput methods. SDA may beincorporated in a microarray-based application, where small volumeamounts (nanoliters) of sample and reagents may be used to amplify HSVtarget DNA and detect the amplification products on a microchip array byperforming multiple SDA assays on a single platform. The primaryadvantage of the SDA method for detecting HSV in a sample is the minimallabor requirement and high-throughput potential since the isothermalamplification process presents significantly fewer technical challengesin design and maintenance of the platform.

The term “target” or “target sequence,” as used herein, refers to HSVnucleic acid sequences, HSV-1 or HSV-2, to be amplified and detected.These include the original HSV nucleic acid sequence to be amplified,the complementary second strand of the original HSV nucleic acidsequence to be amplified, and either strand of a copy of the originalHSV sequence which is produced by the amplification reaction. Thesecopies serve as amplifiable targets since they contain copies of thesequence to which the amplification primers anneal. Copies of the targetsequence which are generated during the amplification reaction arereferred to as amplification products, amplimers or amplicons. The HSV-1and HSV-2 target sequences are located in the Glycoprotein G (US4) geneof the HSV-1 and HSV-2 genomic sequences. The HSV-1 target sequence islocated between bases 555 and 680 of the consensus sequence of FIG. 1.The HSV-2 target sequence is located between bases 867 and 990 of theconsensus sequence of FIG. 6. The Glycoprotein G (US4) gene is locatedbetween position 136,744 and 137,460 of the HSV-1 genomic sequence andbetween positions 137,878 to 139,977 of the HSV-2 genomic sequence ofFIGS. 2 and 7, respectively.

As used herein, an “amplification primer” is a primer that anneals to atarget sequence and can be extended by amplification. The region of theamplification primer that binds to the target sequence is the targetbinding sequence. Amplification techniques include, but are not limitedto, Strand Displacement Amplification (SDA), including thermophilic SDA(tSDA), Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR), insitu hybridization, Self-Sustaining Sequence Replication (3SR), RollingCircle Amplification (RCA), Nucleic Acid Sequence Based Amplification(NASBA), And Transcription Mediated Amplification (TMA).

In one embodiment, an amplification primer may be used in a StrandDisplacement Amplification (SDA) method. The amplification primercomprises at the 3′ end, a target binding sequence portion which bindsto the HSV target sequence, and at the 5′ end, a portion that does notbind or anneal to the target sequence. The portion of the SDAamplification primer that does not bind the target sequence alsocomprises a tail and a recognition site for a restriction endonucleaseupstream of the target binding sequence as described in U.S. Pat. No.5,455,166 and U.S. Pat. No. 5,270,184, incorporated herein by reference.This recognition site is specific for a restriction endonuclease whichwill nick one strand of a DNA duplex when the recognition site ishemimodified, as described by Walker, et al. (1992. Proc. Natl. Acad.Sci. USA 89:392-396 and 1992 Nucl. Acids Res. 20:1691-1696). The tail isupstream of the restriction endonuclease recognition site sequence andfunctions as a polymerase repriming site when the remainder of theamplification primer is nicked and displaced during SDA. The reprimingfunction of the tail sustains the SDA reaction and allows synthesis ofmultiple amplicons from a single target molecule. The length andsequence of the tail are generally not critical and can be routinelyselected and modified.

One embodiment of the invention is based on the target binding sequenceconferring target specificity on the amplification primer, where itshould be understood that the target binding sequences exemplified inthe present invention may also be used in a variety of other ways fordetection of HSV. For example, the target binding sequences disclosedherein may alternatively be used as hybridization probes for directdetection of HSV, either without prior amplification or in apost-amplification assay. Such hybridization methods are well known inthe art and typically employ a detectable label associated with orlinked to the target binding sequence to facilitate detection ofhybridization. Furthermore, Tables 1 and 2 list primer sequences (SEQ IDNOs: 5-25 and 3647, respectively) containing a target binding sequencewhich is indicated by capitalization and underlining. These targetbinding sequences may be used as primers in amplification reactionswhich do not require additional specialized sequences (such as, PCR) orappended to the appropriate specialized sequences for use in NASBA, insitu hybridization, TMA, 3SR, other transcription based amplificationprimers which require an RNA polymerase promoter linked to the targetbinding sequence of the primer, or any other primer extensionamplification reactions. These amplification methods which requirespecialized non-target binding sequences in the primer are necessary forthe amplification reaction to proceed and typically serve to append thespecialized sequence to the target. For example, the restriction enzymerecognition site is necessary for exponential amplification to occur inSDA (see U.S. Pat. Nos 5,455,166 and 5,270,184). Amplification primersfor Self-sustained Sequence Replication (3SR) and Nucleic AcidSequence-Based Amplification (NASBA), in contrast, comprise an RNApolymerase promoter near the 5′ end. (3SR assays are described inGuatelli et al., 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878) Thepromoter is appended to the target binding sequence and serves to drivethe amplification reaction by directing transcription of multiple RNAcopies of the template. Linking such specialized sequences to a targetbinding sequence for use in a selected amplification reaction is routineand well known to one of ordinary skill in the art.

In contrast, amplification methods such as PCR, which do not requirespecialized sequences at the ends of the target, generally employamplification primers consisting of only target binding sequence. Fordetection purposes in these other amplification methods, the primers maybe detectably labeled as understood by the skilled artisan.

As nucleic acids do not require complete complementarity in order toanneal, one skilled in the art would understand that the probe andprimer sequences disclosed herein may be modified to some extent withoutloss of utility as HSV-1- and HSV-2-specific primers and probes. Theterm “homology” refers to a degree of complementarity. There may bepartial homology or complete homology, wherein complete homology isequivalent to identity. A partially complementary sequence that at leastpartially inhibits an identical sequence from hybridizing to a targetnucleic acid is referred to as “substantially homologous.” Theinhibition of hybridization of the completely complementary sequence tothe target sequence may be examined using a hybridization assay (e.g.,Southern or Northern blot, solution hybridization and the like) underconditions of low stringency. A substantially homologous sequence orprobe will compete for and inhibit the binding (L e., the hybridization)of a completely homologous sequence or probe to the target sequenceunder conditions of low stringency. Nonetheless, conditions of lowstringency do not permit non-specific binding; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction.

As will be understood by those of skill in the art, the stringency ofannealing may be altered in order to identify or detect identical orrelated polynucleotide sequences. As will be further appreciated by theskilled practitioner, the melting temperature, T_(m), may beapproximated by the formulas as known in the art, depending on a numberof parameters, such as the length of the primer or probe in number ofnucleotides, or annealing buffer ingredients and conditions (see, forexample, T. Maniatis et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1982 and J.Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y., 1989; Current Protocols inMolecular Biology, Eds. F. M. Ausubel et al., Vol. 1, “Preparation andAnalysis of DNA”, John Wiley and Sons, Inc., 1994-1995, Suppls. 26, 29,35 and 42; pp. 2.10.7-2.10.16; G. M. Wahl and S. L. Berger (1987;Methods Enzymol. 152:399-407); and A. R. Kimmel, 1987; Methods ofEnzymol. 152:507-511). As a general guide, T_(m) decreases approximately1° C.-1.5° C. with every 1% decrease in sequence homology. Temperatureranges may vary between about 50° C. and 62° C., but the amplificationprimers may be designed to be optimal at 52° C. However, temperaturesbelow 50° C. may result in primers lacking specificity, whiletemperatures over 62° C. may result in no hybridization. A furtherconsideration when designing amplification primers is the guanine andcytosine content. Generally, the GC content for a primer may be about60-70%, but may also be less and can be adjusted appropriately by oneskilled in the art. The hybridizing region of the target bindingsequence may have a T_(m) of about 42° C.-48° C. Annealing complementaryand partially complementary nucleic acid sequences may be obtained bymodifying annealing conditions to increase or decrease stringency (i.e.,adjusting annealing temperature or salt content of the buffer). Suchminor modifications of the disclosed sequences and any necessaryadjustments of annealing conditions to maintain HSV-1 and HSV-2specificity require only routine experimentation and are within theordinary skill in the art.

The amplification primers designed for detection of HSV-1 and HSV-2target sequences are identified in Tables 1 and 2 as SEQ ID NOs: 7-18and 38-43, respectively. These amplification primers are designed suchthat the target binding sequence anneals to a segment of the highlyhomologous consensus Glycoprotein G (US4) gene region (see, FIGS. 1-2and 6-7). HSV target binding sequence regions within the amplificationprimers that anneal to or are complementary to HSV target DNA sequences,are underlined and capitalized (see, Tables 1 and 2). The remaining 5′portion of the SDA detection primer sequences comprises the BsoBIrestriction endonuclease recognition site (RERS) (as indicated inlowercase italics) that is required for the SDA reaction to proceed, aswell as, a generic non-target-specific 5′ tail end sequence.

HSV-1 and HSV-2 amplification primers of SEQ ID NOs: 7-8 and 38,respectively, are left hand (“first”) S1 amplification primers, and SEQID NOs: 9-18 and 39-43, respectively, are right hand (“second”) S2amplification primers. For amplification purposes, a pair of HSVamplification primers of a specific type may be used alone (i.e., oneHSV-1 left amplification primer and one HSV-1 right amplificationprimer) or in combination (i.e., one HSV-1 SDA left primer and two HSV-1SDA right primers), such that there is at least one left and right handprimer pair in the reaction. Multiple amplification primers may be usedto amplify several regions of the target sequence. The concentrations ofprimers may be adjusted appropriately, such that when an HSV-1 firstamplification primer is used as the sole first amplification primer at aconcentration of 500 nM, two HSV-1 right amplification primers may beused in conjunction, each have a concentration of 250 nM.

The term “extension product” generally refers to the sequence producedby extending a primer or target sequence using an enzyme, such aspolymerase. In one embodiment, hybridization of an amplification primerand extension of the amplification primer by polymerase using the HSVtarget sequence as a template produces an amplification primer extensionproduct.

A “bumper primer” or “external primer” is a primer that anneals to atarget sequence upstream of the amplification primer such that extensionof the bumper primer displaces the downstream amplification primer andits extension product. As used herein, the term “bumper primer” refersto a polynucleotide comprising an HSV target binding sequence. Usefulbumper primers are identified in Tables 1 and 2 as SEQ ID NOs: 23-25 and46-47, respectively. The left or first HSV-1 and HSV-2 bumper primersare SEQ ID NOs: 23 and 46, respectively, while the right or second HSV-1and HSV-2 bumper primers are SEQ ID NOs: 24-25 and 47, respectively.Bumper primers are derived from conserved regions of sequence that flankthe amplification primers at a position upstream of the amplificationprimers that is sufficiently close to the target binding site of theamplification primer to allow displacement of the amplification primerextension product after extension of the bumper primer. For example, the5′ end of the HSV-1 first bumper primer of SEQ ID NO: 23 (HSV1GGLB1.0)is located at base 137,256 of the HSV-1 genomic sequence (FIG. 2). The5′ end of the HSV-1 second bumper primer of SEQ ID NO: 25 (HSV1GGRB1.1)is located at base 137,382 of the HSV-1 genomic sequence (FIG. 2).During the initial round of SDA, the bumper primers hybridize to the HSVtarget sequence and displace by polymerase extension, the downstreamamplification primer extension products, resulting in the generation ofa single-stranded DNA that may undergo further rounds of replicationand/or exponential amplification.

The term “assay probe” refers to any nucleic acid used to facilitatedetection or identification of a nucleic acid. For example, in anembodiment of the present invention, assay probes are used for detectionor identification of HSV nucleic acids. Detector probes, detectorprimers, capture probes and primers as described below are examples ofassay probes.

In particular, “detector probes” useful in detecting and identifyingspecific HSV- types are labeled or tagged. The detectable label of thedetector probe is a moiety that may be detected either directly orindirectly, indicating the presence of the target nucleic acid sequence.For direct detection, the assay or detector probe may be tagged with aradioisotope and detected by autoradiography or tagged with afluorescent moiety and detected by fluorescence as known in the art.Alternatively, the assay probes may be indirectly detected by labelingwith additional reagents that enable the detection. Indirectlydetectable labels include, for example, chemiluminescent agents, enzymesthat produce visible or colored reaction products, and aligand—detectably labeled ligand binding partner, where a ligand (e.g.,haptens, antibodies, or antigens) may be detected by binding to labeledligand-specific binding partner.

For detection of the amplification products, amplification primerscomprising the target binding sequences disclosed herein may be labeledas is known in the art, or labeled detector primers comprising thedisclosed target binding sequences may be used in conjunction with theamplification primers as described in U.S. Pat. No. 5,547,861; U.S. Pat.No. 5,928,869; U.S. Pat. No. 5,593,867; U.S. Pat. No. 5,550,025; U.S.Pat. No. 5,935,791; U.S. Pat. No. 5,888,739; U.S. Pat. No. 5,846,726 forreal-time homogeneous detection of amplification. Such detector primersmay comprise a directly or indirectly detectable sequence which does notinitially hybridize to the target but which facilitates detection of thedetector primer once it has hybridized to the target and been extended.For example, such detectable sequences may be sequences which contain arestriction site, or sequences which form a secondary structure whichbrings fluorophore and quencher moieties in close proximity, such as,but not limited to hairpin and g-quartet sequences, or linear sequenceswhich are detected by hybridization of their complements to a labeledoligonucleotide (sometimes referred to as a reporter probe) as is knownin the art. Alternatively, the amplification products may be detectedeither in real-time or post-amplification through the use ofintercalating dyes or post-amplification by hybridization of a probeselected from any of the target binding sequences disclosed herein whichfall between a selected set of amplification primers.

Terminal and internal labeling methods are known in the art and may beused to link the donor and acceptor dyes at their respective sites inthe detector primer. Examples of 5′-terminal labeling methods include a)periodate oxidation of a 5′-to-5′ coupled ribonucleotide followed byreaction with an amine-containing label, b) condensation ofethylenediamine with a 5′-phosphorylated polynucleotide followed byreaction with an amine-reactive label, and c) introduction of analiphatic amine substituent using an aminohexyl phosphite reagent insolid-phase DNA synthesis followed by reaction with an amine-reactivelabel. Labels may also be linked to synthetic DNA oligonucleotides atspecific locations using special aliphatic amine-containing nucleotidephosphoramidite reagents. Selection of an appropriate method for linkingthe selected labels to the detector primer and performing the linkingreactions are routine in the art.

Another embodiment utilizes a detector primer that hybridizes to aspecific target sequence resulting in the necessity for multipledetector primers depending on the target sequence being detected.However, an embodiment for the detection and identification of thespecific HSV-type uses the Universal detection system, which is modifiedfrom the real-time SDA detection method described by Nadeau, et al.(1999). The Universal detection system permits the use of the same pairof fluorescent detector primers for multiple assays, offering severaladvantages such as cost, time, and reduced technical complexity.

“Signal” or “adapter” primers have a target binding portion thathybridizes to the HSV target sequence and a tail portion that is genericand does not bind to the HSV target sequence. Adapter primers are usedin conjunction with detector primers for Universal detection. Thedetector primer hybridizes to the tail portion (i.e., the non-targetbinding sequence) of the complement adapter primer. Signal or adapterprimers are designed to hybridize to regions of the target sequence thatlie at least partially in the intervening region between the first andsecond amplification primers so that the signal or adapter primers aredisplaced during the amplification reaction. HSV-1 and HSV-2 signal oradapter primers having SEQ ID NOs: 19-22 and 44-45 are shown in Tables 1and 2, respectively.

The detector probe may be a “universal detector primer” or “detectorprimer” which has a 5′ tail end portion that is detectably labeled and a3′ end portion which binds to the complement adapter primer tailsequence. Generally, the 3′ end of the detector primer does not containsequences with any significant complementarity to the HSV or InternalAmplification Control (IAC) target sequence. The detector primer alsohas a restriction enzyme recognition site at the 5′ end.

Briefly, this Universal detection system can be used simultaneously andin the same reaction container as the SDA method for amplification. TheUniversal detection system involves the target-dependent extension of anunlabeled adapter primer. The adapter primer comprises an HSV-1 or HSV-2target specific 3′ sequence and 5′ generic tail and is exemplified inSEQ ID NOs: 19-22 and SEQ ID NOs: 44-45, and its complement,respectively. The adapter primer hybridizes to the amplified HSV targetsequence downstream of the S1 amplification primer. DNA polymeraseextends from the 3′ ends of the adapter primer and the S1 amplificationprimer, where the extension of the amplification primer displaces theadapter primer extension product. The S2 amplification primer anneals tothe adapter primer extension product. DNA polymerase extends the 3′ endof the S2 amplification primer, producing a double-stranded moleculecomprising the adapter primer extension product and its complement, andhas a nickable restriction enzyme recognition site. Nicking refers tobreaking the phosphodiester bond of only one of two strands in a DNAduplex. A corresponding restriction enzyme nicks the double-strandedmolecule at the restriction enzyme recognition site creating a 5′portion comprising a short nicked tail and a 3′ portion comprising along nicked complement adapter primer extension product. Nicking therestriction enzyme site with a corresponding restriction enzyme, suchas, BsoBI enzyme, and extending the strand from the nicked sitedisplaces a single-stranded copy of the adapter primer complement. DNApolymerase extends the 3′ end of the nicked tail, thereby displacing thesingle-stranded nicked complement adapter primer extension product. TheS1 amplification primer extension product and extended HSV targetsequence may be further amplified exponentially by SDA. The displacedcomplement adapter primer extension product is then captured by adetector primer, where the 3′ end of the detector primer anneals to the5′ portion of the complement adapter primer extension product. Thedetector primer comprises a detectable label and detects targetsequence. DNA polymerase extension from the 3′ ends of the detectorprimer and the complement adapter primer extension product results inopening the hairpin, if present, producing a double-stranded detectionmolecule comprising a detector primer extension product and itscomplement. Each strand comprises a cleavable restriction enzymerecognition site, which when cleaved separates the donor and quencherdyes, separating the fluorophore and the quencher moieties, andgenerating target-specific fluorescence. Due to the separation, thequencher is no longer capable of suppressing the fluorescence emitted bythe fluorophore. Complete cleavage of the double-stranded detectorprimer restriction enzyme recognition site increases the fluorescentsignal by separating the fluorophore and quencher.

In an embodiment of the invention, detector primers may be tagged forfluorescence detection with a fluorescent donor moiety (or fluorophore)and a quencher moiety where each moiety flanks the restriction enzymerecognition site. Tables 1 and 2 show detector primer sequences havingSEQ ID NOs: 30-35. In Universal detection, the detector primers fordetecting target sequence are generally used in conjunction with adapterprimers. Detector primers that are labeled with a donor dye, rhodamine(ROX™), and a quencher dye, P-(dimethyl aminophenylazo) benzoic acid(DABCYL™) having SEQ ID NOs: 30-33 are used for HSV target sequencedetection in an embodiment of the invention. Other donor and quencherdye pairs may be readily selected for use in the SDA by one skilled inthe art, such that the quencher dye sufficiently absorbs thefluorescence emitted by the donor dye. For example, the donor andquencher dyes are readily detected and differentiated by absorption atdifferent wavelengths. Depending on the donor and quencher dyes, thequencher dye may act as a quencher in one instance and as a donor dye inother.

In this embodiment, the detector primer of SEQ ID NOs: 30-35 has a donorand quencher dye pair separated by a restriction enzyme recognition sitelocated at the 5′ end of the detector primer. Furthermore, the detectorprimer of SEQ ID NO: 30 has a sequence comprising a hairpin structuresequence located between the donor and quencher moities, where therestriction enzyme recognition site lies therein. The hairpin structurebrings the two dyes in close proximity such that the fluorescenceemitted by the donor dye is suppressed by the quencher dye. However, thedetector primers of SEQ ID NOs: 31-35 have a linear sequence between thetwo dyes which is short enough in length for the quencher to absorb anyfluorescence emitted by the fluorophore.

Many donor/quencher dye pairs known in the art are useful in embodimentsof the present invention. These include, but are not limited to,fluorescein (FAM™; Glen Research; Sterling, Va.)/rhodamine (ROX™;Molecular Probes; Eugene, Oreg.); ROX™/P-(dimethyl aminophenylazo)benzoic acid (DABCYL™; Glen Research); FAM™/DABCYL™; fluoresceinisothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC);FITC/Texas Red™ (Molecular Probes); FITC/N-hydroxysuccinimidyl1-pyrenebutyrate (PYB); FITC/eosin isothiocyanate (EITC);N-hydroxysuccinimidyl 1-pentanesulfonate (PYS)/FITC; FITC/Rhodamine X;and FITC/tetramethylrhodamine (TAMRA). The selection of a particulardonor/quencher pair is not critical.

However, for energy transfer quenching mechanisms, it is only necessarythat the emission wavelengths of the donor fluorophore overlap theexcitation wavelengths of the quencher, i.e., there must be sufficientspectral overlap between the two dyes to allow efficient energytransfer, charge transfer or fluorescence quenching. ROX™ has anEMmax=608 nm and FAM™ has an EMmax of 520 nm. One skilled in the artwould be knowledgeable in selecting the appropriate donor and quencherdye pair. P-(dimethyl aminophenylazo) benzoic acid (DABCYL™) is anon-fluorescent quencher dye which effectively quenches fluorescencefrom an adjacent fluorophore, e.g., FAM™ or5-(2′-aminoethyl)aminonaphthalene (EDANS). Certain donor/quencher pairsare exemplified in this disclosure; however, others will be apparent tothose skilled in the art and are also useful in the invention. Any dyepair which produces fluorescence quenching in the detector primers ofthe invention are suitable for use in the methods of the invention,regardless of the mechanism by which quenching occurs. Non-limitingexamples of other quenchers include Black Hole Quencher™ (BiosearchTechnologies, Inc.; Novato, Calif.) and Iowa Black™ (Integrated DNATechnologies, Inc.; Corralville, Iowa).

Fluorescence is measured during the course of the nucleic acidamplification reaction to monitor the accumulation of specificamplification products. The fluorescent signal is proportional to theamount of specific amplicon produced. In the presence of HSV targetnucleic acid sequence, fluorescence will increase. In the absence oftarget, fluorescence will remain consistently low throughout thereaction. An increase in fluorescence or a failure of fluorescence tochange substantially indicates the presence or absence of HSV targetsequence, respectively.

The fluorescence of the samples is typically measured over time todetermine whether a sample contains HSV DNA. In one embodiment,fluorescence may be monitored for 60 passes over the course of one hour.Briefly, approximately every minute, data are collected regarding theamount of fluorescence measured in the sample container, a correctionvalue (if necessary), and calibrators for each column. Data may beanalyzed using the “MOTA” (Metric Other Than Acceleration) method ofexpressing results in terms of the area under a curve of a graph. Thegraph measures the number of passes (X-axis) versus relative fluorescentunits (Y-axis) (see, FIG. 3). The greater the MOTA area, the morefluorescence generated and the more efficient the detection of amplifiedproducts.

Yet another embodiment uses a Passes After Threshold (PAT) algorithm,which is shown in FIG. 4, and is particularly developed for use with theBD ProbeTec™ ET System. Similar to MOTA, a higher PAT score indicates amore efficient SDA reaction. When using the PAT algorithm, the time atwhich the background corrected signal of fluorescence intensity crossesa predetermined threshold is designated as T3 (“Time-To-Threshold”).This graph also measures the number of passes to relative fluorescentunits. The same T3 threshold value is used for every sample. The PATscore is equal to 60 minus the T3 value. Negative samples do not achievethe minimum threshold of fluorescence and are therefore assigned a PATvalue of zero. Positive samples have PAT values greater than 0,preferably between 1 and 60, more preferably between 40-55, depending onthe assay and target level. Lower T3 scores and corresponding higher PATvalues correlate with a more efficient SDA. The PAT algorithm utilizesonly the region of the amplification curve that is the mostreproducible. As a result, the PAT algorithm method minimizesdiscernable differences between wells or samples, and is more precisethan other methods of comparison between detectors. PAT can be performedautomatically by the BD ProbeTec™ ET System. The BD ProbeTec™ ETprintout provides a PAT score and a reportable result.

In yet a further embodiment, an “internal amplification control” (“IAC”)may be incorporated into the present method to verify negative resultsand to identify potential inhibitory specimens or to facilitatequantification of organism load in a sample, such as but not limited toviruses, bacteria, and fungi. For diagnostic applications, simultaneousamplification and detection of two different DNA sequences, i.e., theHSV target sequence and the IAC target sequence, enable the use of anIAC. The “IAC target sequence” or “IAC sequence” is similar to the HSVtarget sequence with the exception that the IAC target sequences of SEQID NOs: 26-27 and of SEQ ID NOs: 48-49 are mismatched by about 5-10bases compared to the HSV-1 and HSV-2 target sequences. These modifiedbases are sufficient to allow specific annealing of IAC adapter primers.

“IAC adapter primers” function similarly to the signal or adapterprimers with the exception that the IAC adapter primers hybridize to an“IAC target sequence” or “IAC sequence” through an IAC target bindingsequence. The IAC adapter primer also has a 5′ tail portion containing ageneric sequence which does not hybridize to the IAC target sequence.Rather a detector primer may hybridize to the tail portion of the IACadapter primer complement. The IAC adapter primers used in the HSV-1 andHSV-2 SDA assays may be selected from SEQ ID NOs: 28-29 and 50-51,respectively, and are useful in the amplification of IAC targetsequences. The IAC target binding sequence located at the 3′ end of theIAC adapter primer differs from the, HSV target sequence sufficientlysuch that the HSV-1 or HSV-2 adapter primers do not hybridize orinterfere with the amplification of the IAC target sequence. In Tables 1and 2, the IAC target binding sequence at the 3′ end of the IAC adapterprimer is indicated by lowercase underlining. The IAC adapter primersare useful in verifying negative results and in monitoring for specimensthat inhibit the reaction. For quantitative SDA, competition forrate-limiting reagents between an IAC and a native target sequence mayalso be useful (Nadeau, et al., 1999 Anal. Biochem. 276: 177-187).

Detector primers of the invention may be used to detect either HSVtarget sequences or IAC target sequences. However, in one embodiment ofthe invention, detector primers used to detect the HSV-1 or HSV-2 targetsequence are those of SEQ ID NOs: 30-33. The detector primers useful indetecting IAC target sequences are those selected from SEQ ID NOs:34-35, where the donor and quencher dye pair is fluorescein (FAM™) andDABCYL™, respectively, and may be referred to herein as “IAC detectorprimers.” One skilled in the art would be knowledgeable in selecting theappropriate detector primers having labels, such that the identificationof the IAC target sequence is distinguishable from the identification ofthe HSV-1 or HSV-2 target sequence. Therefore, the detector primers usedin the detection of HSV target sequence and IAC target sequence may beexchanged such that detector primers of SEQ ID NOs: 30-33 and may beused in the detection of IAC target sequences and SEQ ID NOs: 34-35 maybe used in the detection of HSV target sequences.

Another embodiment of the invention relates to assaying multiple samplessimultaneously in a high-throughput process. Samples include, but arenot limited to those collected from cerebral spinal fluid (CSF), genitallesions, oral lesions, mucosal lesions, ocular specimens, dermalspecimens, rectal swabs, vaginal swabs, vaginal secretions, urine,peripheral blood leukocytes, and tissue (such as from a brain biopsy).The samples may be assayed in plates, slides, wells, dishes, beads,particles, cups, strands, chips, and strips. In one embodiment, themethods are performed in 96 micro-well plates in a format consistentwith that used in the BD ProbeTec™ ET CT/GC Amplified DNA Assay. Themethod is performed in a dried micro-well format, where the driedcomposition comprises all of the primers and probes necessary forcarrying out SDA detection of HSV-1 or HSV-2 for use in simultaneouslyassaying multiple samples.

Assays detecting the presence of a selected target sequence according tothe methods of the invention may be performed in solution or on a solidphase. Real-time or endpoint homogeneous assays in which the detectornucleic acid functions as a primer are typically performed in solution.Hybridization assays using the detector primers of the invention mayalso be performed in solution (e.g., as homogeneous real-time assays)but are also particularly well-suited to solid phase assays forreal-time or endpoint detection of target. In a solid phase assay,detector oligonucleotides may be immobilized on the solid phase (e.g.,beads, membranes or the reaction vessel) via internal or terminal labelsusing methods known in the art. For example, a biotin-labeled detectoroligonucleotide may be immobilized on an avidin-modified solid phasewhere it will produce a change in fluorescence when exposed to thetarget under appropriate hybridization conditions. Capture of the targetin this manner facilitates separation of the target from the sample andallows removal of substances in the sample which may interfere withdetection of the signal or other aspects of the assay.

The primers and probes used for detecting and identifying HSV-1 targetsequence are listed in Table 1. The specific HSV target bindingsequences are underlined and capitalized, while the restriction enzymeendonuclease sites are indictated in lower case italics. For the IACadapter primers, the IAC target binding sequence is indicated by lowercase underlining. All primers are listed in the 5′→3′ direction.

TABLE 1 PRIMER SEQUENCES FOR AMPLIFICATION AND DETECTION OF SEQ IDHERPES SIMPLEX VIRUS 1 DNA NO:PCR AMPLIFICATION PRIMERS FOR THE HSV-1 TARGET SEOUENCE PCRL1.0GCGGAATTCGACCCTTGGTTCC 5 PCRR1.0 GCGGGATCCCCAACCACCACAC 6LEFT (FIRST) AMPLIFICATION PRIMER HSV1GGLP1.0 ACCGCATCGAATGACTGTctcgggCTGTTCTCGTTCCTC 7 HSV1GGLP1.1 ACCGCATCGAATGACTGTctcggg CTGTTCTCGTTCCT 8RIGHT (SECOND) AMPLIFICATION PRIMER HSV1GGRP1.0 CGATTCCGCTCCAGACTTctcgggCACCAATACACAAAAA 9 HSV1GGRP1.1 CGATTCCGCTCCAGACTTctcggg CAACAATACACACAAA10 HSV1GGRP2.0 CGATTCCGCTCCAGACTTctcggg CACCAATACACAAAAAC 11 HSV1GGRP2.1CGATTCCGCTCCAGACTTctcggg CAACAATACACACAAAC 12 HSV1GGRP3.0CGATTCCGCTCCAGACTTctcggg CACCAATACACAAAAACG 13 HSV1GGRP3.1CGATTCCGCTCCAGACTTctcggg CAACAATACACACAAACG 14 HSV1GGRP4.0CGATTCCGCTCCAGACTTctcggg CAATACACAAAAACGAT 15 HSV1GGRP4.1CGATTCCGCTCCAGACTTctcggg CAATACACACAAACGAT 16 HSV1GGRP4.2CGATTCCGCTCCAGACTTctcggg CAATACACACAAATGAT 17 HSV1GGRP5.2CGATTCCGCTCCAGACTTctcggg AAGGTGTGGATGAC 18 ADAPTER PRIMER HSV1GGAD1.0ACGTTAGCCACCATACGGATCCGTCATCCACACCTTATC 19 HSV1GGAD2.1ACGTTAGCCACCATACGGATGGACACCCTCTTCGTCGTC 20 HSV1GGAD3.0ACGTTAGCCACCATACTTGAGGACACCCTCTTCGTCGTC 21 HSV1GGAD3.1ACGTTAGCCACCATACTTGAGGACACCCTCTTCGTCG 22 LEFT (FIRST) BUMPER PRIMERHSV1GGLB1.0 GACGCCTCAACATAC 23 RIGHT (SECOND) BUMPER PRIMER HSV1GGRB1.0GTGTGTCGCCATCG 24 HSV1GGRB1.1 AGGTGTGTCGCCAT 25 IAC TARGET SEQUENCEHSV1IAC8.1 CTGTTCTCGTTCCTCACTGCCTCCCCCGCCCTGGACACCCTC 26TTGCTGCTGAGCACCGTCATCCACACCTT HSV1IAC8.7CTGTTCTCGTTCCTCACTGCCTCCCCCGCCCTGGACACCCTC 27TGTTCATCTAGCACCGTCATCCACACCTT IAC ADAPTER PRIMER HSV1IACAD8.1ACTGATCCGCACTAACGACTggacaccctcttgctgctg 28 HSV1IACAD8.7ACTGATCCGCACTAACGACTggacaccctctgttcatct 29 DETECTOR PRIMER TBD10.2 D/R(DABCYL)-TAGCGcccgagCGCT-(ROX)- 30 ACGTTAGCCACCATACGGAT TBD15 D/R(DABCYL)-TGcccgagT-(ROX)-ACGTTAGCCACCATACGGAT 31 TBD16 (D/R)(DABCYL)-TcccgagT-(ROX)-ACGTTAGCCACCATACGGAT 32 MPC.DR(DAB CYL)-TCcccgagT-(ROX)-ACGTTAGCCACCATACTTGA 33 MPC2.FD(FAM)-TCcccgagT-(DABCYL)-ACTGATCCGCACTAACGACT 34 AltD8 (F/D)(FAM)-AcccgagT-(DABCYL)-AGCTATCCGCCATAAGCCAT 35

The primers and probes used for detecting and identifying HSV-2 targetsequence are listed in Table 2.

TABLE 2 PRIMER SEQUENCES FOR AMPLIFICATION AND DETECTION OF SEQ IDHERPES SIMPLEX VIRUS 2 DNA NO:PCR AMPLIFICATION PRIMERS FOR THE HSV-2 TARGET SEQUENCE HSV2PCRLGCGGAATTCATTCTTGGGCCGCT 36 HSV2PCRR GCGGGATCCACGTAACGCACGCT 37LEFT (FIRST) AMPLIFICATION PRIMER HSV2GGLP1.0 ACCGCATCGAATGACTGTctcgggCTGTTCTGGTTCCTA 38 RIGHT (SECOND) AMPLIFICATION PRIMER HSV2GGRP1.0CGATTCCGCTCCAGACTTctcggg CGACCAGACAAACGAA 39 HSV2GGRP1.1CGATTCCGCTCCAGACTTctcggg ACCAGACAAACGAAC 40 HSV2GGRP1.2CGATTCCGCTCCAGACTTctcggg CGACCAGACAAACGAAC 41 HSV2GGRP2.0CGATTCCGCTCCAGACTTctcggg AACGCCGCCGTGT 42 HSV2GGRP5.2CGATTCCGCTCCAGACTTctcggg CCGTGTGGATGGT 43 ADAPTER PRIMER HSV2GGAD1.0ACGTTAGCCACCATACGGATCCACCATCCACACGGCGGC 44 HSV2GGAD2.0ACGTTAGCCACCATACTTGATGCTCTAGATATCCTCTTTATCAT 45LEFT (FIRST) BUMPER PRIMER HSV2GGLB1.0 CACACCCCAACACAT 46RIGHT (SECOND) BUMPER PRIMER HSV2GGRB1.0 TTGTGCTGCCAAGG 47IAC TARGET SEQUENCE HSV2-IAC 5.2A1CTGTTCTGGTTCCTAACGGCCTCCCCTGCTCTAGATATCCTCT 48TTACTACCAGCACCACCATCCACACGG HSV2-IAC 5.2A2CTGTTCTGGTTCCTAACGGCCTCCCCTGCTCTAGATATCCTCT 49TAACTACCAGCACCACCATCCACACGG IAC ADAPTER PRIMER HSV2GG IACACTGATCCGCACTAACGACTtgctctagatatcctctttactac 50 ADA1.0 HSV2GG IACACTGATCCGCACTAACGACTtgtctagatatcctcttaactac 51 ADA2.0 DETECTOR PRIMERTBD10.2 D/R (DABCYL)-TAGCGcccgagCGCT-(ROX)- 30 ACGTTAGCCACCATAC GGATTBD15 D/R (DABCYL)-TGcccgagT-(ROX)-ACGTTAGCCACCATACGGAT 31 TBD16 (D/R)(DABCYL)-TcccgagT-(ROX)-ACGTTAGCCACCATACGGAT 32 MPC.DR(DABCYL)-TCcccgagT-(ROX)-ACGTTAGCCACCATACTTGA 33 MPC2.FD(FAM)-TCcccgagT-(DABCYL)-ACTGATCCGCACTAACGACT 34 ALTD8 (F/D)(FAM)-AcccgagT-(DABCYL)-AGCTATCCGCCATAAGCCAT 35

The nucleic acid primers of the present invention are designed based ona consensus sequence generated by analyzing the Glycoprotein G (US4)sequence region of the HSV gene for various strains. (See, FIGS. 1 and6; Tables 1 and 2). Also shown are bumper primers, adapter primers, anddetector primers for use in the SDA and universal detection methods. Thedesigned HSV-1 primers specifically amplify an HSV-1 target sequencethat is recognized in all strains as exemplified in Table 4. The HSV-2primers are designed to specifically amplify an HSV-2 target sequencethat is recognized in all strains as exemplified in Table 7. Since thehomology between the HSV-1 and HSV-2 target sequences is about 90%, theprimers are carefully designed to specifically distinguish between HSV-1and HSV-2. Also contemplated in the invention, are sequences thatsubstantially homologous to the target binding sequences and primerscontaining such substantially homologous target binding sequences listedin Tables 1 and 2.

In one embodiment of the present invention, an HSV-1 target region isfirst selected from the complete HSV-1 genomic sequence of Human HSV-1,strain 17 (NCBI accession no. X14112) having 152,261 bases in length.The glycoprotein “US4” gene is located at 136,744-137,460 bases. TheHSV-1 Left bumper primer (HSV1LB1.0) (5′ end) is located at nucleic acid137,256. The HSV-1 Right bumper primer (HSV1RB1.1) (5′ end) is locatedat nucleic acid 137,382. Primers for all HSV-1 SDA systems are locatedwithin these bumper primer coordinates.

Another embodiment of the invention relates to the complete HSV-2 genomesequence of Human HSV-2, strain HG52 (NCBI accession no. Z86099) having154,746 bases in length. The glycoprotein G “US4” gene is located at137,878-139,977 bases. The HSV-2 Left bumper primer (HSV2LB1.0) (5′ end)is located at position 139,773. The HSV-2 Right bumper primer(HSV2RB1.0) (5′ end) is located at position 139896. Primers for allHSV-2 SDA systems are located within the bumper primer coordinates.

PCR amplification primers are designed for cloning the HSV target DNAinto a plasmid vector. The HSV-1 and HSV-2 PCR amplification primers ofSEQ ID NOs: 5-6 and SEQ ID NOs: 36-37, respectively, are complementaryto highly conserved target sequence regions of the HSV genome. The PCRamplification primers amplify an HSV target sequence region comprising aDNA fragment of the Glycoprotein G (US4) gene of HSV. The amplifiedfragment of the herpes simplex virus (HSV) genome containing the HSVtarget region is directionally cloned into a plasmid vector containingconvenient restriction enzyme sites. Although the HSV fragment may becloned into any plasmid vector as is understood by the skilled artisan,in one embodiment of the invention, the amplified HSV-1 and HSV-2fragments are cloned into the Escherichia coli plasmid vectors, pUC19(Genbank/EMBL Accession No. L09137) and pUC18 (Genbank/EMBL AccessionNo. L09136), respectively, using PCR amplification primers specific tothe selected HSV target regions. The HSV fragment is referred to as theHSV target stock. The target HSV DNA may be quantified using thePicoGreen® double stranded DNA Quantitation Assay (Molecular. Probes,Inc.). The presence of “L” or “R” in the primer name listed in Tables 1and 2 indicates “left” or “right” primers, respectively, when used inamplification reactions.

In one embodiment of the invention, PCR amplification primers SEQ IDNOs: 5-6 and 36-37 initially amplify a 152 and 254 base pair fragment ofthe Glycoprotein G gene of the HSV-1 and HSV-2 gene, respectively. TheHSV-1 and HSV-2 Left PCR primer of SEQ ID NOs: 5 and 36, respectively,are each designed with an EcoRI restriction enzyme site. The HSV-1 andHSV-2 Right PCR primer of SEQ ID NOs: 6 and 37, respectively, each havea BamHI restriction enzyme site. This fragment is then positionallycloned into the pUC plasmid vector. The exemplified plasmid vector ispUC19 and pUC18 for HSV-1 and HSV-2, respectively, which haverestriction enzyme sites EcoRI and BamHI. After purification andlinearization by restriction enzyme digestion, the HSV target fragmentis then exponentially amplified using HSV amplification primers andbumper primers.

The target binding sequences and primers of the invention are useful innucleic acid amplification. In one embodiment, the primers areparticularly useful in strand displacement amplification (SDA). This isan isothermal method of nucleic acid amplification in which extension ofprimers, nicking of hemimodified restriction endonucleaserecognition/cleavage site, displacement of single-stranded extensionproducts, annealing of primers to the extension products (or theoriginal target sequence) and subsequent extension of the primers occurconcurrently in a reaction mixture. Furthermore, SDA allows for targetsequence replication in excess of 10¹⁰ fold in less than 15 minutes.Whereas, in PCR, the steps of the reaction occur in separate phases orcycles as a result of temperature cycling in the reaction. ThermophilicStrand Displacement Amplification (tSDA) is performed essentially as theconventional SDA method described herein and by Walker, et al. (1992,Proc. Natl. Acad. Sci USA. USA 89:392-396 and 1992, Nucl. Acids Res.20:1691-1696) with substitution of the thermostable polymerase andthermostable restriction endonuclease. The temperatures may be adjustedto the higher temperature appropriate for the substituted enzymes.

An alternative method of detecting HSV amplification products oramplified target sequence may be by detecting a characteristic size bypolyacrylamide or agarose gel electrophoresis, where the agarose isstained with ethidium bromide. The amplified products generated usingthe HSV-1 or HSV-2 amplification primers may also be detected byquantitative hybridization, or equivalent techniques for nucleic aciddetection known to one skilled in the art of molecular biology (Sambrooket al, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, Cold Spring, N.Y. (1989)).

The primers listed in Tables 1 and 2 are useful in the detection andidentification of HSV-1 and HSV-2 in a sample. As used herein, the S1and S2 amplification primers represent the first and secondamplification primers, respectively; while the B1 and B2 bumper primersrepresent the first and second bumper primers, respectively. Briefly, inthe SDA method, the S1 amplification primer hybridizes to asingle-stranded HSV target sequence. Just upstream or 5′ of the S1amplification primer, a first bumper primer, B1, hybridizes to thesingle-stranded HSV target sequence. DNA polymerase extends the 3′ endsof the B1 bumper primer and the S1 amplification primer, where theextension of the B1 bumper primer eventually displaces the S1 SDAextension product. The S1 SDA extension product is captured by the S2amplification primer and B2 bumper primer which anneals upstream of theS2 amplification primer. DNA polymerase extends the 3′ ends of the S2SDA and B2 bumper primers, where the extension of the B2 bumper primerdisplaces the downstream S2 SDA extension product. The S1 amplificationprimer anneals to the displaced S2 amplification primer extensionproduct and DNA polymerase extends the 3′ end of the hybridized S1amplification primer, producing a double-stranded molecule having the S2amplification primer extension product and its complement strand. Eachstrand has a nickable restriction enzyme recognition site at either end.Upon addition of the corresponding restriction enzyme, the modified DNAstrand, containing a thiolated cytosine, is nicked forming a shortnicked tail and a long extension product 3′ of the nick site. DNApolymerase extends the short nicked tail from the 3′ end of the shortnicked tail in a 5′→3′ direction displacing the single-stranded longextension product. Briefly, the nicked tail of the S2 amplificationprimer extension product and the nicked tail of its complement displacethe single-stranded nicked S2 amplification primer extension product andsingle-stranded nicked complement S2 amplification primer extensionproduct, respectively. In one embodiment, BsoBI enzyme is used to nickand cleave or cut each strand having a sequence of SEQ ID NOs: 52-53 and54-55, respectively. The nick sites, indicated below, are incorporatedinto the amplification primer sequence and require ahemi-phosphorothiolated recognition sequence (dCsTP, thiolatedcytosine). Although a nick site, SEQ ID NO: 53 is prone todouble-stranded cleavage even in the presence of dCsTP and is not apreferred sequence in designing nickable amplification primers.

Nick Sites: 5′-CTCGGG-3′ (SEQ ID NO: 52) and 5′-CCCGGG-3′(SEQ ID NO: 53) Cut Sites: 5′-CTCGAG-3′ (SEQ ID NO: 54) and 5′-CCCGAG-3′(SEQ ID NO: 55)

In a further embodiment of the invention, a detector probe is useful indetecting the HSV target sequence. The S1 amplification primer and adetector primer specific for the HSV target sequence may be used, wherethe detector primer has a an HSV target binding sequence. DNA polymeraseextends from the 3′ ends of the S1 primer and the detector primer.Extension of the S1 primer displaces the downstream detector primerextension product into solution, where it is captured and hybridizes toa complementary S2 amplification primer. DNA polymerase extends the 3′end of the S2 amplification primer and opens up the secondary structureof the detector primer forming a double-stranded restriction enzyme siteand separating the two dyes (fluorophore and quencher pair) to such adistance as to disable the quenching ability of the quencher and togenerate fluorescence. Additional fluorescence is produced by cleavingthe restriction enzyme recognition site and further separating thefluorophore and quencher.

Enzymes useful in the SDA method are those that create a single-strandednick in a hemi-phosphorothioated recognition sequence, where theincorporation of phosphorothioated nucleotides does not prevent furtherrounds of nicking and repair. Non-limiting examples of enzymes thatpossess these characteristics include: HincII, BsoBI, AvaI, NciI, andFnu4HI. Useful DNA polymerases are those that initiate DNA synthesis atthe single-stranded nick site, incorporate phosphorothioated nucleotidesinto the extending nucleic acid chain, and displace strands without5′→3′ exonuclease activity. Cleavage refers to the breaking of thephosphodiester bond of the double-stranded or single-stranded DNA.Non-limiting examples of DNA polymerases that exhibit thosecharacteristics include: exonuclease-deficient Klenow andexonuclease-deficient fragments of Bst polyermase and Bca polymerase.Although other DNA polymerases and restriction enzymes are suitable forSDA (Walker et al. Proc. Natl. Acad. Sci USA, Vol. 89, pp. 392-396,January 1992, Applied Biological Sciences), exo-Bst polymerase and BsoBIwere chosen for their thermal characteristics and compatibility with oneanother. In one embodiment of the invention, BsoBI restrictionendonuclease recognition sites are used and designated in italics (see,Tables 1 and 2). It will be readily apparent that the HSV target bindingsequences may be used alone to amplify the HSV target in reactions whichdo not require specialized sequences or structures (e.g., PCR) and thatother specialized sequences required by amplification reactions otherthan SDA (e.g., an RNA polymerase promoter) may be substituted in thesystem, for example for the RERS-containing sequence described herein.

The target stock may then be amplified in the presence of amplificationprimers, alone or in combination with bumper primers, signal/adapterprimers for universal detection, and a universal detector primer. For anamplification reaction, at least one pair comprising one “left”amplification primer is selected and one “right” amplification primer isselected to amplify each strand of the HSV target stock sequence. Inaddition to the left and right amplification primers, in the SDAreaction, one left and right bumper primer pair is initially used.Furthermore, for detection, a signal/adapter primer and a detectionprimer is selected and used to detect and identify the HSV targetsequence.

Several HSV systems that specifically amplify and detect either HSV-1 orHSV-2 DNA are embodied in the present invention. For example, HSV-1systems may include the following primers: HSV1GGLP1.1, HSV1GGRP5.2,HSV1GGAD2.1, HSV1GGLB1.0, HSV1GGRB1.1, and TBD16 (D/R) or alternatively,HSV1GGLP1.1, HSV1GGRP5.2, HSV1GGAD3.0 or HSV1GGAD3.1, HSV1GGLB1.0,HSV1GGRB1.1, MPC.DR, HSV1IAC AD8.1 or HSV1IACAD8.7, MPC2.FD. In anotherembodiment, HSV-2 systems using various combinations of primers arelisted in Table 3. Other combinations of primers are contemplatedhowever, one skilled in the art would be knowledgeable in combining theprimers in order to detect HSV-1 or HSV-2 in a sample. The primers maybe selected from those listed in Tables 1 and 2, and tested instatistically designed experiments in order to identify HSV-1 or HSV-2in a sample. Alternatively, primers that are specific for HSV-1 or HSV-2and substantially homologous to those listed in Tables 1 and 2 may alsobe used in the detection of HSV-1 or HSV-2 target sequences.

TABLE 3 HSV-2 SDA SYSTEM DESIGNS HSV2 SDA SYSTEM PRIMERS USED IN SYSTEMHSV2GG 1.0 HSV2GGRP1.0 Right amplification primer HSV2GGLP1.0 Leftamplification primer HSV2GGRB1.0 Right Bumper primer HSV2GGLB1.0 LeftBumper primer HSV2GGAD1.0 Adapter primer HSV2GG 1.1 HSV2GGRP1.1 Rightamplification primer HSV2GGLP1.0 Left amplification primer HSV2GGRB1.0Right Bumper primer HSV2GGLB1.0 Left Bumper primer HSV2GGAD1.0 Adapterprimer HSV2GG 1.2 HSV2GGRP1.2 Right amplification primer HSV2GGLP1.0Left amplification primer HSV2GGRB1.0 Right Bumper primer HSV2GGLB1.0Left Bumper primer HSV2GGAD1.0 Adapter primer HSV2GG 2.0 HSV2GGRP2.0Right amplification primer HSV2GGLP1.0 Left amplification primerHSV2GGRB1.0 Right Bumper primer HSV2GGLB1.0 Left Bumper primerHSV2GGAD2.0 Adapter primer HSV2GG 5.2 HSV2GGRP5.2 Right amplificationprimer HSV2GGLP1.0 Left amplification primer HSV2GGRB1.0 Right Bumperprimer HSV2GGLB1.0 Left Bumper primer HSV2GGAD2.0 Adapter primer

For commercial convenience, amplification primers for specific detectionand identification of nucleic acids may be packaged in the form of akit. Generally, such a kit contains at least one pair of HSVamplification primers. Reagents for performing a nucleic acidamplification reaction may also be included with the target-specificamplification primers, for example, buffers, additional primers,nucleotide triphosphates, enzymes, etc. The components of the kit arepackaged together in a common container, optionally includinginstructions for performing a specific embodiment of the inventivemethods. Other optional components may also be included in the kit,e.g., a primer tagged with a label suitable for use as an assay probe,and/or reagents or means for detecting the label.

In one embodiment of the invention, a kit is provided that comprises afirst amplification primer or S1 SDA amplification primer, and a secondamplification primer or S2 SDA amplification primer. The kit may furthercomprise a first B1 bumper primer and second B2 bumper primer; anadapter primer; a detector primer; and optionally, reagents forsimultaneously detecting an Internal Amplification Control (IAC) targetsequence, including IAC adapter primers and an IAC target sequence. Thekit may comprise of primers specifically for HSV-1 or HSV-2, or the kitmay comprise of primers directed to both HSV-1 and HSV-2, where oneskilled in the art would understand that amplification reactions todetect and identify HSV-1 utilize the HSV-1 primers, and to detect andidentify HSV-2 utilize HSV-2 primers. In order to identify whether asample contains HSV-1 or HSV-2 DNA, primers for HSV-1 and HSV-2 shouldnot be mixed.

In yet another embodiment, the kit and primers of the invention may beused to detect and diagnose whether a clinical sample contains HSV-1 orHSV-2 DNA. The clinical sample may be amplified and detected using theSDA amplification primers, or may be used in an SDA reaction furthercomprising bumper primers, adapter primers, and detector primers. In anembodiment of the invention, IAC adapter primers may be used as aninternal amplification control for the reactions, in addition topositive and negative controls for HSV-1 or HSV-2. One skilled in theart would understand, from reading the description herewith and from thegeneral methods and techniques in the art, how to make and use theprimers for the detection and identification of HSV-1 and HSV-2 in asample.

Furthermore, in a commercial embodiment, a composition comprising theprimers of the invention and reagents for SDA may be provided in a driedor liquid format. The composition is more stable and easily manipulatedwhen in a dried format. The dried composition may be added orpre-treated to a solid phase such as a microliter plate, microarray, orother appropriate receptacle, where the sample and SDA buffer need onlybe added. This format facilitates assaying multiple samplessimultaneously and is useful in high-throughput methods. In anembodiment of the invention, the BD ProbeTec™ ET instrument may be used.

It is to be understood that a nucleic acid according to the presentinvention which consists of a target binding sequence and, optionally,either a sequence required for a selected amplification reaction or asequence required for a selected detection reaction may also includecertain other sequences which serve as spacers, linkers, sequences forlabeling or binding of an enzyme, or other uses. Such additionalsequences are typically known to be necessary to obtain optimum functionof the nucleic acid in the selected reaction.

The contents of all patents, patent applications, published PCTapplications and articles, books, references, reference manuals andabstracts cited herein are hereby incorporated by reference in theirentirety to more fully describe the state of the art to which theinvention pertains.

As various changes may be made in the above-described subject matterwithout departing from the scope and spirit of the present invention, itis intended that all subject matter contained in the above description,or defined in the appended claims, be interpreted as descriptive andillustrative of the present invention. Many modifications and variationsof the present invention are possible in light of the above teachings.

EXAMPLES

The Examples herein are meant to exemplify the various aspects ofcarrying out the invention and are not intended to limit the scope ofthe invention in any way. The Examples do not include detaileddescriptions for conventional methods employed, such as in theconstruction of vectors or the insertion of cDNA into such vectors. Suchmethods are well known to those skilled in the art and are described innumerous publications, for example, J. Sambrook and D. W. Russell,Molecular Cloning: a Laboratory Manual, 3^(rd) Edition, Cold SpringHarbor Laboratory Press, USA, (2001).

Example 1 Cloning of HSV-1 Glycoprotein-G Strand DisplacementAmplification Target Region

PCR was performed on DNA from the HSV-1 (strain ATCC: VR-539) using thePCR amplification primers of SEQ ID NO: 5 and SEQ ID NO:6 identified inTable 1. These primers were designed to amplify a 152 base pair fragmentwithin the Glycoprotein G (US4) gene of HSV-1. PCR amplified DNA wasinserted into a pUC19 plasmid vector (Gibco BRL; Grand Island, N.Y.).The recombinant clone was named HSV1GG Plasmid #1. Plasmid #1 DNA waspurified and linearized by digestion with BamH1 restriction enzyme. TheDNA was then purified using QIAquick (Qiagen, Inc.; Valencia, Calif.)spin columns and quantified by analysis with fluorescent Picogreen®reagent. Dilutions of the target HSV-1 DNA for future experiments wereprepared in water containing 10 ng/μl human DNA. Specific HSV-1 straindilutions and the results of HSV-1 detection in each dilution are shownin FIG. 5. A “plus” symbol indicates the presence of HSV-1 in thesample; a “minus” symbol indicates the absence of HSV-1 in the sample;and a question mark indicates the suspected contamination of the sample.All strains were positive at 1:10 dilution of Stock, except sampleO-2526. At a 1:1,000 dilution of the stock, 20 of the 23 strains werepositive. At a 1:100,000 dilution of the stock, 15 of 23 strains werepositive.

Example 2 Amplification of Cloned HSV-1 DNA

As an initial step in the assay for detecting HSV-1 DNA, an SDA systemfor amplification of HSV-1 DNA was developed using the target nucleicacid method described in Example 1. The analytical senstivity of the DNAamplification assay was estimated using dilutions of the cloned plasmid.Eight replicate SDA reactions were performed at each target level. Theseeight reactions were equivalent to 100, 50, 25, 12.5, 6.25 and 0 copiesof double stranded DNA per reaction. Amplification was conducted at 52°C. using a BD ProbeTec™ ET instrument (BD Diagnostic Systems; Sparks,Md.) with 50 nM each of HSV1GGLB1.0 and HSV1GGRB1.0 bumper primers, 100nM of HSV1GGLP1.0 left amplification primer, 500 nM HSV1GGRP1.0 rightamplification primer, 250 nM HSV1GGAD1.0 adapter primer, 500 nM TBD10.2D/R detector primer. The sequences of these primers are listed inTable 1. Final buffer conditions were as follows: 143 mM Bicine, 82 mMKOH, 25 mM KiPO₄, 12.5% DMSO, 5 mM magnesium acetate, 500 mM2′-deoxycytosine-5-o-(1-thiotriphosphate) (dC_(s)TP), 100 nM each ofdATP, dGTP, and dTTP, 100 ng/μl BSA, approximately 12 units of Bstpolymerase and approximately 30 units of BsoBI restriction endonuclease.

Fluorescence was monitored for 60 passes over the course of one hour.Results were expressed in terms of area under the curve or “MOTA” score.Positive MOTA scores can be readily determined by routineexperimentation. For the purpose of the present invention, MOTA scoresgreater than or equal to 3500 were considered “positive.” The lowestlevel of HSV 1GG target DNA at which the assay yielded 100% positiveresults was 100 copies per reaction. Seven of eight replicates (87.5%)were also positive at fifty copies of target DNA per reaction.

Example 3 Detection of Herpes Simplex 1 Virus Particles by SDA

To verify the ability of the primers and probes of the invention todetect HSV-1, SDA was performed on four strains of HSV-1 obtained fromAmerican Type Culture Collection (ATCC; Manassas, Va.), 10 strainsobtained from Quest Diagnostic (Baltimore, Md.) and fourteen untyped HSVsamples from Ohio State University (OSU).

The untyped strains of HSV were characterized by amplifying a region ofthe DNA polymerase gene by PCR. One set of amplification primers wasdesigned to amplify the same region in both HSV-1 and HSV-2. The twotypes of virus were distinguishable by the presence of an Apalrestriction endonuclease recognition site in the HSV-2 PCR fragment thatis not present in PCR products generated from strains of HSV-1. Whenincubated with the Apal restriction enzyme, the HSV-2-derivedamplification products are cleaved into two shorter fragments whilethose obtained from HSV-1 remain intact. Restricted fragments wereresolved on agarose gel electrophoresis with appropriate controls.

The concentrations of the viral stocks used to evaluate the presence orabsence of HSV in an SDA system were not known. The viral stocks werediluted 1:10 in phosphate buffered saline and 10 μL of this suspensionwas tested by SDA. Results are shown in Table 4. All strains of HSV-1were detected using the HSV-1 amplification primers demonstrating theability of disclosed primers and probes to detect strains of HSV-1 froma diversity of sources. Of the previously untyped 14 strains of HSVtyped by Apal restriction digest, nine were determined to be HSV-1, fourwere determined to be HSV-2 and one did not amplify by PCR (see, Tables4 and 5).

TABLE 4 STRAINS OF HERPES SIMPLEX 1 VIRUS TESTED BY SDA APA1 HSV- HSVSAMPLE GEL HSV-1GG 1GG TYPE # COMMENTS RESULTS (MOTA) (PAT) HSV-1 OSUpreviously HSV-1 16970 45.91 0-2021 untyped HSV-1 OSU previously HSV-1105400 50.07 0-450 untyped HSV-1 OSU previously HSV-1 156280 52.430-1010 untyped HSV-1 OSU previously HSV-1 154715 51.96 0-2526 untypedHSV-1 OSU previously HSV-1 149720 54.73 D-8-1973 untyped HSV-1 OSUpreviously HSV-1 152600 54.56 7-370 untyped HSV-1 OSU previously HSV-1175610 54.77 0116-3 untyped HSV-1 OSU 1136 previously HSV-1 165410 53.36untyped HSV-1 OSU A.P. previously HSV-1 225340 54.62 untyped HSV-1 ATCCATCC HSV-1 148960 54.78 260 VR HSV-1 ATCC ATCC HSV-1 248080 54.73 VR-733HSV-1 ATCC ATCC HSV-1 153760 54.46 VR-735 HSV-1 ATCC ATCC HSV-1 17386054.47 VR-539 HSV-1 Clin1 Quest Diagnostics HSV-1 250570 54.33 HSV-1Clin2 Quest Diagnostics HSV-1 222590 54.52 HSV-1 Clin3 Quest DiagnosticsHSV-1 126780 51.51 HSV-1 Clin4 Quest Diagnostics HSV-1 262540 54.63HSV-1 Clin5 Quest Diagnostics HSV-1 180530 54.44 HSV-1 Clin6 QuestDiagnostics HSV-1 12750 35.12 HSV-1 Clin7 Quest Diagnostics HSV-1 11550050.31 HSV-1 Clin8 Quest Diagnostics HSV-1 184130 54.29 HSV-1 Clin9 QuestDiagnostics HSV-1 197860 52.98 HSV-1 Clin10 Quest Diagnostics HSV-1160660 50.99

Example 4 Analytical Sensitivity of the SDA Method

To determine the limit of detection of the HSV-1 assay using the primersand probes disclosed in the present invention, SDA reactions wereperformed on dilutions of cloned target nucleic acid and serialdilutions of viral particles. The stock of viral particles wasenumerated by electron microscopy (Electron Microscopy Bioservices).Sixteen replicates were tested at each target level.

To verify the sensitivity and specificity of the assay, 23 stains ofHSV-1 from various geographical locations were tested at a 1:10, 1:1,000and 1:100,000 dilution of the organism stock. The titer of the samplesfrom the previously un-typed strains and from Quest Diagnostics wasunknown. The titer of the two stocks of HSV-1 from ATCC wereapproximately as follows: VR260, 1.5×10⁴ TCID/μL and VR-539, 2.0×10⁵TCID/μl. Results are shown in FIG. 5. All strains were positive at 1:10dilution of the stock suspension, except Strain #0-2526. Of the 23strains, 20 tested positive at 1:1000 dilution of the stock, and 15strains tested positive at dilution of 1:100,000.

Example 5 Specificity of SDA for HSV-1 DNA

Sixteen strains of HSV-2 were tested with the HSV1GG SDA system. Tenmicroliters of each suspension of HSV-2 dilution were added perreaction. One of the 17 stocks tested positive with the HSV1GG system.The results are shown in Table 5. In addition, 23 other microorganismswere tested using the primers and probes of the disclosed inventivemethod. These microorganisms included bacteria, yeast and other viruseslikely to be encountered in clinical specimens. None of the organismstested positive for HSV-1. Results are shown in Table 6.

TABLE 5 SPECIFICITY OF HSV-1 PRIMERS AND PROBES HSV- SAMPLE APAI GELHSV-1GG 1GG # COMMENTS INTERPRETATION (MOTA) (PAT) 0-2053 previouslyHSV-2 680 0 untyped 0-1753 previously No Product 90 0 untyped D-8575previously HSV-2 390 0 untyped C5 (S?) previously HSV-2 150 0 untypedJuly-67 previously HSV-2 740 0 untyped ATCC ATCC HSV-2 30 0 VR-734 ATCCATCC HSV-2 100 0 VR-540 Clin11 Quest HSV-2 410 0 Diagnostics Clin12Quest HSV-2 20 0 Diagnostics Clin13 Quest HSV-2 290 0 Diagnostics Clin14Quest HSV-2 320 0 Diagnostics Clin15 Quest HSV-2 40 0 Diagnostics Clin16Quest HSV-2 210 0 Diagnostics Clin17 Quest HSV-2 10 0 Diagnostics Clin18Quest HSV-2 40 0 Diagnostics Clin19 Quest HSV-1* 145050 53.38Diagnostics Clin20 Quest HSV-2 220 0 Diagnostics *Clin19 was typed asHSV-2 by Quest and typed HSV-1 by ApaI analysis.

TABLE 6 SPECIFICITY OF HSV-1 PRIMERS AND PROBES HSV-1GG HSV-1GG OrganismStrain # (MOTA) SDA (PAT) Adenovirus-5 ABI 74-070 180 0 Candida albicansATCC 44808 0 0 Cryptococcus neoformans ATCC 36556 60 0 Cytomegalovirus(AD-169) ABi 68-125 10 0 Enterovirus (Echovirus-11) ABi 74-084 20 0Epstein-Barr virus SIGMA 240 0 104HO854 Escherichia coli ATCC 11775 0 0Fusobacterium nucleatum ATCC 25586 0 0 Group B Streptococcus ATCC 12386130 0 Hameophilus influenzae ATCC 33533 320 0 Listeria moncytogenes ATCC7644 40 0 Mycoplasma pneumoniae ATCC 63-030 300 0 Neisseria meningitidisATCC 13077 10 0 Propioibacterium acnes ATCC 6919 0 0 Pseudomonasaeruginosa ATCC 27853 170 0 Resp. Synctial virus ABi 74-093 180 0Staphylococcus. Aureus ATCC 25923 0 0 Staphylococcus epidermidis ATCCE155 0 0 Streptococcus mitis ATCC 6249 10 0 Streptococcus mutans ATCC25175 20 0 Streptococcus pneumoniae ATCC 6303 0 0 Streptococcus pyogenesATCC 19615 0 0 Rhiniovirus Clin 74 250 0 ATCC—American Type CultureCollection; ABi—Advanced Biotechnologies, Inc.

Example 6 Cloning of HSV-2 Glycoprotein-G SDA Target Region

PCR was performed on DNA from the HSV-2 strain ATCC VR-540 using theprimers HSV2PCRR and HSV2PCRL with an annealing temperature of 69° C.These primers amplify a 254 base pair fragment within the Glycoprotein G(US4) gene of HSV-2. Amplified DNA was inserted into a pUC18 plasmidvector (Invitrogen). The recombinant clone was dubbed pHSV2-NT #9-1.Plasmid DNA was purified and linearized by digestion with BamHIrestriction enzyme. The DNA was purified using QIAGEN QIAquick spincolumns and quantified by analysis with fluorescent Picogreen® reagent.Dilutions of the target DNA for future experiments were prepared inwater containing 7 ng/μL salmon sperm DNA.

Example 7 Amplification of Cloned HSV-2 DNA

The analytical sensitivity of the DNA amplification assay was estimatedusing dilutions of the cloned plasmid. Eight replicate SDA reactionswere performed at each target level using systems 2.0 and 5.2. Theseeight reactions were equivalent to 500, 250, 100, 50, 25, 10 and 0copies of double stranded DNA per reaction. Amplification was conductedat 52° C. using a BD ProbeTec™ ET instrument with 50 nM each ofHSV2GGLB1.0 and HSV2GGRB1.0, 100 nM of HSV2GGLP1.0, 500 nM HSV2GGRP2.0or 5.2, 250 nM HSV2GGAD1.0, 500 nM MPC.D/R. The sequences of theseprimers are listed in Table 2. Final buffer conditions were as follows:71 mM Bicine, 56.6 mM KOH, 23.9 mM KPO4, 15.4% DMSO, 5 mM magnesiumacetate, 500 mM 2′-deoxycytosine-5-o-(1-thiotriphosphate) (dCsTP), 100nM each of dATP, dGTP and dTTP, 100 μg/μL BSA, approximately 3.515 unitsof Bst polymerase and approximately 30 units of BsoBI restrictionendonuclease.

Fluorescence was monitored for 60 passes over the course of one hour.Results were expressed in terms of area under the curve or “MOTA” score,and as PAT scores (Passes After Threshold). For the purpose of thepresent invention, MOTA scores greater than or equal to 3500 and PATscores greater than 0 were considered “positive.” The lowest level ofHSV2GG target DNA at which the assay yielded 100% positive results wasat 50 copies per reaction for both systems 2.0 and 5.2. Seven of eightreplicates were positive for system 2.0 at 25 copies, and six of eightwere positive for system 5.2 at 25 copies.

Example 8 Detection of HSV-2 Virus Particles by SDA

To verify the ability of the primers and probes of the invention todetect HSV-2, SDA was performed on two strains of HSV-2 obtained fromAmerican Type Culture Collection (ATCC), nine strains obtained fromQuest Diagnostic (Baltimore, Md.) and five strains from Ohio StateUniversity (OSU) typed as HSV-2 by ApaI analysis.

The strains from OSU were characterized by amplifying a region of theDNA polymerase gene by PCR. One set of PCR primers was designed toamplify the same region in both HSV-1 and HSV-2. The two types of viruswere distinguishable by the presence of an ApaI restriction endonucleaserecognition site in the HSV-2 PCR fragment that is not present in PCRproducts generated from strains of HSV-1. When incubated with the ApaIrestriction enzyme, the HSV-2 derived PCR products are cleaved into twoshorter fragments while those obtained from HSV-1 remain intact.Restricted fragments were resolved on agarose gel electrophoresis withappropriate controls.

The concentrations of the viral stocks used to evaluate the SDA systemwere not known. The viral stocks were diluted 1:10 in phosphate bufferedsaline and 10 μL of this suspension was tested in SDA. Results are shownin Table 7. All strains of HSV-2 were detected using the amplificationprimers from systems 1.0, 2.0 and 5.2, demonstrating the ability ofdisclosed primers and probes to detect strains of HSV-1 from a diversityof sources.

TABLE 7 STRAINS OF HSV-2 TESTED IN THE HSV2GG SYSTEMS DILUTION HSV2GGHSV2GG HSV2GG FROM 1.0 HSV2GG 2.0 HSV2GG 5.2 HSV2GG STRAIN STOCK MOTA1.0 PAT MOTA 2.0 PAT MOTA 5.2 PAT OSU 0- 1:10 5460 34 103902 52.2 10433452.6 2053 OSU D- 1:10 59950 50 123676 52.6 129392 52.7 8575 OSU C5 1:1034230 47 96845 52.4 103945 52.6 OSU 7- 1:10 135900 53 88932 52.4 8642652.6 2667 ATCC 1.58E+9 TCID/μL 173220 54 108624 52.4 105501 52.6 VR-734ATCC 1.58E+4 TCID/μL 183190 55 131364 52.5 128644 52.6 VR-540 Quest 1:10157930 52 83950 52.4 86787 52.5 Clin 11 Quest 1:10 127670 51 129929 52.3122640 52.4 Clin 12 Quest 1:10 136710 51 103652 52.2 101533 52.4 Clin 13Quest 1:10 135660 53 106922 52.2 110146 52.5 Clin 14 Quest 1:10 8440 38110309 51.1 141752 52.2 Clin 15 Quest 1:10 157960 53 125560 52.1 13323252.4 Clin 16 Quest 1:10 203550 53 85670 51.4 81501 52.2 Clin 17 Quest1:10 64690 48 153890 52.1 154132 52.4 Clin 18 Quest 1:10 225400 54142087 52.4 119663 52.5 Clin 20 OSU = Ohio State University; ATCC =American Type Culture Collection; Quest = Quest Diagnostics

Example 9 Specificity of SDA for HSV-2 DNA

Twenty-five strains of HSV-1 were tested with HSV2GG SDA systems 1.0,2.0 and 5.2. Ten microliters of each suspension of the HSV-1 dilutionwere added per reaction. None of the 25 strains were detected by theHSV-2 systems. Results are shown in Table 8. In addition, a panel ofother microorganisms was tested using the primers and probes of thedisclosed invention method. These microorganisms included bacteria,yeast and other viruses likely to be encountered in clinical specimens.None of the organisms tested positive for HSV-2. Results are shown inTable 9.

TABLE 8 SPECIFICITY FOR HSV-2 STRAINS AGAINST HSV-1 STRAINS IN THEHSV2GG SYSTEMS Dilution HSV2 HSV2 HSV2 HSV2 HSV2 HSV2 HSV-1 from GG 1.0GG 1.0 GG 2.0 GG 2.0 GG 5.2 GG 5.2 Strain Stock MOTA PAT MOTA PAT MOTAPAT 0-2021 1:10 250 0 2059 0 433 0 0-450 1:10 150 0 500 0 632 0 0-10101:10 580 0 1063 0 1733 0 OSU0-2526 1:10 330 0 988 0 816 0 OSU0-1753 1:10250 0 16 0 4 0 OSUD-8- 1:10 90 0 1 0 433 0 1973 OSU7-370 1:10 110 0 6120 102 0 OSU0116-3 1:10 290 0 25 0 45 0 OSU1136 1:10 390 0 88 0 61 0OSUA.P. 1:10 470 0 No data No data No data No data ATCC 2.8E+6 TCID/μL610 0 656 0 858 0 260VR ATCC VR- 15.8 TCID/μL 1270 0 No data No data Nodata No data 733 ATCC VR- 15.8 TCID/μL 130 0 0 0 1008 0 735 ATCC VR-1000 vp/μL 480 0 926 0 1429 0 539 Quest Clin 1 1:10 990 0 1011 0 64 0Quest Clin 2 1:10 190 0 0 0 68 0 Quest Clin 3 1:10 790 0 419 0 75 0Quest Clin 4 1:10 310 0 No data No data No data No data Quest Clin 51:10 580 0 311 0 212 0 Quest Clin 6 1:10 370 0 0 0 0 0 Quest Clin 7 1:10380 0 369 0 0 0 Quest Clin 8 1:10 520 0 1576 0 62 0 Quest Clin 9 1:10770 0 2300 0 1138 0 Quest 1:10 440 0 1956 0 1500 0 Clin10 Quest Clin1:10 640 0 587 0 480 0 19 OSU = Ohio State University; ATCC = AmericanType Culture Collection; Quest = Quest Diagnostics

TABLE 9 SPECIFICITY FOR HSV-2 AGAINST VARIOUS BACTERIA, VIRUSES ANDYEASTS USING THE HSV2GG SYSTEMS HSV2 HSV2 HSV2 HSV2 HSV2GG GG 1.0 GG 1.0HSV2 GG GG 2.0 GG 5.2 5.2 Organism MOTA PAT 2.0 MOTA PAT MOTA PATAdenovirus-5 50 0 0 0 581 0 Candida albicans 530 0 0 0 246 0Cryptococcus 120 0 1344 0 1436 0 neoformans Cytomegalovirus 140 0 3 0 10 (AD-169) Enterovirus 680 0 491 0 15 0 (Echovirus-11) Epstein-Barrvirus 530 0 No data No data No data No data Escherichia coli 20 0 1974 069 0 Fusobacterium 0 0 801 0 1560 0 nucleatum Group B 180 0 0 0 387 0Streptococcus Haemophilus 340 0 618 0 1675 0 influenzae Pseudomonas 5000 183 0 1303 0 aeruginosa Respiratory 350 0 4 0 236 0 Synctial VirusStaphylococcus 30 0 885 0 551 0 aureus Staphylococcus 10 0 84 0 831 0epidermidis Streptococcus 190 0 8 0 226 0 mitis Streptococcus 510 0 2940 1813 0 mutans Streptococcus 60 0 404 0 2006 0 pneumoniae Streptococcus0 0 1063 0 3149 0 pyogenes Listeria 470 0 984 0 0 0 monocytogenesMycoplasma 530 0 16 0 408 0 pneumoniae Neisseria 300 0 1661 0 134 0meningitides Propionibacterium 40 0 1559 0 1022 0 acnes Rhinovirus 20 0No data No data No data No data Rhinovirus 74 0 0 No data No data Nodata No data Rhinovirus 114 60 0 No data No data No data No data HIV-1430 0 1335 0 2795 0

1. A polynucleotide comprising an HSV-2 target binding sequenceconsisting of the HSV-2 target binding sequence of any one of SEQ IDNOs:38, 43 and
 45. 2. The polynucleotide of claim 1, which comprises theHSV-2 target binding sequence of SEQ ID NO:38.
 3. The polynucleotide ofclaim 1, which is SEQ ID NO:38.
 4. The polynucleotide of claim 1, whichcomprises the HSV-2 target binding sequence of SEQ ID NO:43.
 5. Thepolynucleotide of claim 1, which is SEQ ID NO:43.
 6. The polynucleotideof claim 1, which comprises the HSV-2 target binding sequence of SEQ IDNO:45.
 7. The polynucleotide of claim 2 or 4, further comprising asequence needed for an amplification reaction to proceed.
 8. Thepolynucleotide of claim 7, further comprising a sequence selected fromthe group consisting of a hairpin, a g-quartet, a restriction enzymerecognition site, an RNA polymerase promoter, and a sequence that bindsto an assay probe.
 9. The polynucleotide of claim 2, 4 or 6, wherein thepolynucleotide is labeled with a detectable label.
 10. Thepolynucleotide of claim 9, wherein the label is a fluorescent moiety.11. The polynucleotide of claim 10, wherein the fluorescent moietycomprises a donor and quencher dye pair selected from the groupconsisting of: fluorescein (FAM)/rhodamine (ROX); FAM/P-(dimethylaminophenylazo) benzoic acid (DABCYL); ROX/DABCYL; fluoresceinisothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC);FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB); FITC/eosinisothiocyanate (EITC); N-hydroxysuccinimidyl 1-pentanesulfonate(PYS)/FITC; FITC/Rhodamine X; and FITC/tetramethylrhodamine (TAMRA). 12.A kit for detecting an HSV-2 target sequence, comprising first, secondand third primers having an HSV-2 target binding sequence consisting ofthe HSV-2 target binding sequence of SEQ ID NOs:38, 43 and 45respectively.
 13. The kit of claim 12, wherein the first primer is SEQID NO:38.
 14. The kit of claim 12, wherein the second primer is SEQ IDNO:43.
 15. The kit of claim 12, wherein the first primer is SEQ ID NO:38and the second primer is SEQ ID NO:43.
 16. The kit of claim 12, whereinthe first, second and/or third primer further comprises a structuralmoiety selected from the group consisting of: a hairpin and g-quartet.17. The kit of claim 12, wherein the first, second or third primerfurther comprises a 5′ restriction enzyme recognition site and adetectable label selected from the group consisting of: a fluorescentmoiety, a radioisotope, a chemiluminescent agent, an enzyme substratecapable of developing a visible reaction product, and aligand-detectably labeled ligand binding partner.
 18. The kit of claim12, wherein the third primer further comprises a detectable label. 19.The kit of claim 18, wherein the detectable label is a fluorescentmoiety.
 20. The kit of claim 19, wherein the fluorescent moietycomprises a donor and quencher dye pair selected from the groupconsisting of: fluorescein (FAM)/rhodamine (ROX); FAM/P-(dimethylaminophenylazo)benzoic acid (DABCYL); ROX/DABCYL; fluoresceinisothiocyanate (FITC)/tetramethylrhodamine isothiocyanate (TRITC);FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate (PYB); FITC/eosinisothiocyanate (EITC); N-hydroxysuccinimidyl 1-pentanesulfonate(PYS)/FITC; FITC/Rhodamine X; and FITC/tetramethylrhodamine (TAMRA). 21.The kit of claim 12, further comprising bumper primers which are SEQ IDNOs:46-47.
 22. The kit of claim 12, wherein the first and second primersare SEQ ID NOS:38 and
 43. 23. The kit of claim 12, wherein the first,second and third primers are in dried form.
 24. A composition comprisingprimers for the detection of an HSV-2 target sequence in a sample by anamplification reaction comprising: (a) a first amplification primerwhich is SEQ ID NO:38; (b) a second amplification primer which is SEQ IDNO:43; (c) a first bumper primer that hybridizes to the HSV-2 targetsequence upstream of the first amplification primer, wherein the firstbumper primer is SEQ ID NO:46; (d) a second bumper primer thathybridizes to a complement of the HSV-2 target sequence upstream of thesecond amplification primer, wherein the second bumper primer is SEQ IDNO:47; and (e) a third primer having an HSV-2 target binding sequenceconsisting of the HSV-2 target binding sequence of SEQ ID NO:45.
 25. Thecomposition of claim 24, wherein the third primer has a detectable labelattached thereto.
 26. The composition of claim 25, wherein thedetectable label is a fluorescent moiety.
 27. The composition of claim26, wherein the fluorescent moiety comprises a donor and quencher dyepair selected from the group consisting of: fluorescein (FAM)/rhodamine(ROX); FAM/P-(dimethyl aminophenylazo) benzoic acid (DABCYL);ROX/DABCYL; fluorescein isothiocyanate (FITC)/tetramethylrhodamineisothiocyanate (TRITC); FITC/N-hydroxysuccinimidyl 1-pyrenebutyrate(PYB); FITC/eosin isothiocyanate (EITC); N-hydroxysuccinimidyl1-pentanesulfonate (PYS)/FITC; FITC/Rhodamine X; andFITC/tetramethylrhodamine (TAMRA).
 28. The composition of claim 24,wherein the first and second amplification primers, the third primer,and the first and second bumper primers are in dried form.
 29. A methodof detecting the presence of HSV-2 target sequence in a sample, saidmethod comprising: (a) adding the sample to a first amplification primerwhich is SEQ ID NO:38; a second amplification primer which is SEQ IDNO:43, and a third detectably labeled primer having an HSV-2 targetbinding sequence consisting of the HSV-2 target binding sequence of SEQID NO:45; producing an amplified HSV-2 nucleic acid product; and (b)detecting the amplified HSV-2 nucleic acid product, wherein thedetection of the amplified product indicates the presence of HSV-2 inthe sample.
 30. The method of claim 29, wherein the amplified HSV-2nucleic acid product is detected by a method selected from the groupconsisting of: universal detection, gel electrophoresis, andquantitative hybridization.
 31. The method of claim 29, wherein (a)further comprises adding the sample to a first bumper primer which isSEQ ID NO:46 and a second bumper primer which is SEQ ID NO:47.
 32. Amethod of detecting an HSV-2 target sequence comprising: (a) amplifyingthe HSV-2 target sequence using a first amplification primer comprisingan HSV-2 target binding sequence consisting of the HSV-2 target bindingsequence of SEQ ID NO:38; (b) amplifying the target sequence using asecond amplification primer comprising an HSV-2 target binding sequenceconsisting of the HSV-2 target binding sequence of SEQ ID NO:43; and (c)detecting the amplified target sequence with a third detectably labeledprimer having an HSV-2 target binding sequence consisting of the HSV-2target binding sequence of SEQ ID NO:45.
 33. The method of claim 32,wherein the amplifying comprises a Strand Displacement Amplification(SDA) reaction.
 34. The method of claim 32, wherein the detection methodis selected from the group consisting of: direct detection, PolymeraseChain Reaction (PCR), in situ hybridization, Transcription MediatedAmplification (TMA), Self-Sustaining Sequence Replication (3SR), RollingCircle Amplification (RCA), Qβ replicase system, and Nucleic AcidSequence Based Amplification (NASBA).
 35. The method of claim 32,wherein the first or second amplification primers further comprises asequence selected from the group consisting of: a hairpin, a g-quartet,a restriction enzyme recognition sequence, and a sequence that binds toan assay probe.
 36. The method of claim 32, wherein the firstamplification primer, the second amplification primer, or both the firstand second amplification primers further comprise at least one sequenceselected from the group consisting of a restriction enzyme recognitionsite and a RNA polymerase promoter.
 37. The method of claim 32, whereinthe third primer is detectably labeled with a fluorescent moiety.